Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus.
Commun Biol
; 7(1): 797, 2024 Jul 02.
Article
en En
| MEDLINE
| ID: mdl-38956406
ABSTRACT
The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Kluyveromyces
/
Sistemas CRISPR-Cas
/
Edición Génica
Idioma:
En
Revista:
Commun Biol
Año:
2024
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Reino Unido